SUBGROUP: Molecular Biophysics 1-Subg Precise Mapping of RNA Tertiary Structure via Nanometer Distance Measurements with Double Electron-Electron Resonance (DEER) Spectroscopy Victoria DeRose 1 , Nak-Kyoon Kim 2 , Michael K. Bowman 3 . 1 University of Oregon, Eugene, OR, USA, 2 University of California-Los Angeles, Los Angeles, CA, USA, 3 University of Alabama, Tuscaloosa, AL, USA. Dynamic RNA structural changes are critical in biology, and a continuing chal- lenge is to develop methods for precision mapping of these structures. Inter-la- bel distance measurements by DEER (Double-Electron Electron Resonance) spectroscopy in conjunction with side-directed RNA spin labeling provide pre- cise nanoscale structural constraints for biomolecules. This technique has been demonstrated in helical and hairpin RNAs, but has rarely been applied to com- plex RNA structures. We have used DEER to monitor a large-scale Mg 2þ -trig- gered RNA folding transition in the Hammerhead ribozyme, a three-helix junction motif that undergoes an inactive-to-active structural change upon addition of Mg 2þ . A distinct increase in the population of ribozymes is ob- served with a short inter-label distance with increasing [Mg 2þ ]. The measured inter-label distance is remarkably consistent with models generated from static crystal structures when it is assumed that the spin labels preferentially localize near to the RNA minor grooves. Data from labels located close to the catalytic core of the ribozyme will probe a putative local structural change in this RNA that may be linked to its catalytic activity. The DEER technique described here can be applied to predict folding of other functional RNA molecules, including those found in complex RNA-protein complexes. 1. Kim, N.-K.; Bowman, M.K.; DeRose, V.J. ‘‘Precise Mapping of RNA Tertiary Structure via Nanometer Distance Measurements with Double Electron-Electron Resonance Spectroscopy (DEER) Spectroscopy’’ J. Amer. Chem. Soc. 2010, 132, 8882-8884. 2-Subg Molecular Insights Into the Organization and Folding Dynamics of Metabolite-Sensing Riboswitches Carlos Penedo 1 , Daniel A. Lafontaine 2 . 1 University of St Andrews, St Andrews, United Kingdom, 2 University of Sherbrooke, Sherbrooke, QC, Canada. Riboswitches are naturally-occurring genetic elements that regulate gene ex- pression usually by binding to specific cellular metabolites. They are composed of two modular domains that correspond to the aptamer and the expression plat- form. The aptamer domain is the most conserved region of the riboswitch as it involved in the specific recognition of the ligand. The expression platform is less conserved and can vary to a high degree in its structure given that it regulates at various biological levels, such as transcription, translation and splicing. Using single-molecule FRET together with a combination of biochemical and biophys- ical assays, we have studied the relationship between aptamer structure, folding and activity in two members of the riboswitch family: the S-adenosylmethionine (SAM) and the adenine riboswitch (A-box).We will propose a folding pathway for each riboswitch aptamer in the presence and absence of their cognate metab- olite and we will demonstrate that the role played by the ligand is fundamentally different in both riboswitch classes. For the adenine aptamer, ligand recognition induces only very subtle changes in the folding dynamics and the local architec- ture of the binding site. On the contrary, binding of the SAM ligand controls the final organization of the SAM aptamer promoting major conformational changes in the aptamer architecture. Our findings provide experimental evidences for a classification of riboswitches according to the degree in which ligand-recogni- tion events control the aptamer folding pathway. 3-Subg Insights Into the Dynamic Personalities of Synthetic Riboswitches by NMR-Spectroscopy Jens Woehnert, Elke Duchardt-Ferner, Sina R. Schmidtke, Amir H. Nasiri, Julia E. Weigand, Beatrix Suess. Johann-Wolfgang-Goethe-University, Frankfurt, Germany. Riboswitches are novel gene regulatory elements found in the 5’- and 3’-un- translated regions of mRNAs. They regulate gene expression upon direct and specific interaction with small molecule ligands. In most riboswitches ligand binding triggers conformational changes on the secondary and/or tertiary struc- ture level which result in changes in gene expression levels. While a lot is known about the structural basis for ligand recognition by riboswitch domains the conformational dynamics of riboswitches which are central to their function is less explored. NMR-spectroscopy in solution is well suited for investigating dynamical systems and for characterising conformational changes. We applied NMR-spectroscopy to two smaller synthetic riboswitches to characterize in de- tail their conformational dynamics in relation to their regulatory functions. 4-Subg Accurate Single-Molecule FRET Studies of Nucleic Acids Using Multi-Parameter Fluorescence Detection Claus A.M. Seidel 1 , Stanislav Kalinin 1 , Simon Sindbert 1 , Hayk Vardanyan 1 , Alessandro Valeri 1 , Suren Felekyan 1 , Sabine Mu ¨ller 2 . 1 Heinrich-Heine University Du ¨sseldorf, Du ¨sseldorf, Germany, 2 Ernst- Moritz-Arndt-Universita ¨t Greifswald, Greifswald, Germany. So far our view of protein function is formed, to a significant extent, by tradi- tional structure determination showing many beautiful static protein structures. Recent experiments by single-molecule and other techniques have questioned the idea that proteins and other biomolecules are static structures. We used multi-parameter fluorescence detection (MFD) to perform smFRET studies of free diffusing biomolecules. We demonstrate that the simultaneous acquisi- tion of most fluorescent parameters by MFD allows for a robust assessment of all possible artefacts involved in single-molecules FRET and offers unsur- passed capabilities regarding the identification and analysis of individual spe- cies present in population of molecules [1]. A toolbox is introduced in order to demonstrate how complications originating from orientation, mobility and position of fluorophores and conformational dynamics [2] have to be taken into account when determining FRET related distances with high accuracy. Al- though static structures are known for many biomolecules, the functions of bio- molecules are governed ultimately by their dynamic character. In this view we give various examples of smFRET experiments of DNA- and RNA-junctions. Their Mg-dependent structural dynamics is studied in detail. These studies show that smFRET studies are valuable tool to complement the structural and dynamic information obtained by X-ray crystallography or NMR spectros- copy. [1] Sisamakis, E., Valeri, A., Kalinin, S., Rothwell, P. J., Seidel, C. A. M.; Accurate single-molecule FRET studies using multiparameter fluorescence detection. Methods in Enzymology 475 (Single Molecule Methods, Part B: Multiparameter, super-resolution, tethering, and force based methods, Ed. Nils Walter) Chapter 18, 455-514 (2010). [2] Kalinin, S., Valeri A., Antonik M., Felekyan, S., Seidel, C. A. M.; Detec- tion of structural dynamics by FRET: A photon distribution and fluorescence lifetime analysis of systems with multiple states. J. Phys. Chem. B. 114, 7983-7995 (2010). 5-Subg The Paths to Specific vs Nonspecific Sequence Recognition Chuanying Chen, B.M. Pettitt. University of Houston, Houston, TX, USA. Our understanding of both nonspecific and specific protein-DNA interaction mechanisms raise questions related to the nature of the transient encounter states, and the structural and energetic features of the protein-DNA binding process. Here we have used the nuclease domain of colicin E7 (N-ColE7) from E. coli to complex with a 12-bp DNA as the model system to draw a pic- ture of how a protein approaches, encounters and associates with DNA. Multi- scale studies using Molecular Dynamics (MD) and Brownian Dynamics (BD) simulations were performed to provide the binding process on multiple length and timescales. We characterize the encounter states and identify the spatial and orientational aspects required for the association by BD simulations. At the atomic length-scales, we investigated the binding process by MD simula- tions. Several intermediate binding states, which have different positions and orientations of protein around DNA in the initial structures are postulated from the MD trajectories. The results facilitate better understanding of se- quence-independent protein-DNA binding landscapes and suggest pathways with favorable intermediate binding states common to both specific and non- specific complexes. 6-Subg RNA Structure, Function, and (Thermo-) Dynamics: A SAXS and Single-Molecule Perspective Jan Lipfert 1 , Daniel Herschlag 2 , Sebastian Doniach 2 , Nynke H. Dekker 1 . 1 Delft University of Technology, Delft, Netherlands, 2 Stanford University, Stanford, CA, USA. Nucleic acids are central to the storage, transmission, and control of genetic in- formation. Their cellular function is dependent not only on sequence, but also on their three-dimensional shape, mechanical properties, and conformational dynamics. Here, we discuss three recent developments that have provided unique insights into these important aspects of DNA and RNA functions. Firstly, small-angle X-ray scattering (SAXS) is emerging as an important tool to study RNA structure. We have demonstrated that SAXS can provide ab initio low-resolution 3D models of RNAs [1]. By taking into account prior Saturday, March 5, 2011 1a